Article pubs.acs.org/biochemistry
Cancer Somatic Mutations Disrupt Functions of the EphA3 Receptor Tyrosine Kinase through Multiple Mechanisms Erika M. Lisabeth,† Carlos Fernandez,† and Elena B. Pasquale*,†,‡ †
Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, United States Department of Pathology, University of California, San Diego, La Jolla, California 92093, United States
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S Supporting Information *
ABSTRACT: The Eph receptor tyrosine kinases make up an important family of signal transduction molecules that control many cellular processes, including cell adhesion and movement, cell shape, and cell growth. All of these are important aspects of cancer progression, but the relationship between Eph receptors and cancer is complex and not fully understood. Genetic screens of tumor specimens from cancer patients have revealed somatic mutations in many Eph receptors. The most highly mutated Eph receptor is EphA3, but its functional role in cancer is currently not well established. Here we show that many EphA3 mutations identified in lung, colorectal, and hepatocellular cancers, melanoma, and glioblastoma impair kinase activity or ephrin ligand binding and/or decrease the level of receptor cell surface localization. These results suggest that EphA3 has ephrin- and kinase-dependent tumor suppressing activities, which are disrupted by somatic cancer mutations.
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motifs contribute to Eph receptor signaling by serving as binding sites for cytoplasmic signaling proteins containing Src-homology 2 (SH2) and phosphotyrosine-binding (PTB) domains. The Eph receptors are expressed in most cell types, including cancer cells and tumor endothelial cells.1,3,10−12 They can have a dual role in tumorigenesis, leading to tumor promotion or tumor suppression depending on the cellular context, the specific receptor involved, and other poorly understood factors. Recent genetic screens of cancer specimens and cell lines have revealed somatic mutations in essentially all Eph receptors in a wide variety of cancer types.1 EphA3 is the Eph receptor found to be most frequently mutated in lung cancer, with 19 missense mutations identified so far and a mutation rate of 6% in a study of 188 lung adenocarcinomas.13−16 EphA3 missense mutations have also been reported in other cancers: six in colorectal cancer,17,18 three in melanoma,19,20 four in glioblastoma,19,21 one in hepatocellular carcinoma,22 two in pancreatic cancer,23 one in head and neck squamous cell carcinoma,24 and four in ovarian cancer25 (Table 1 of the Supporting Information). The 40 different mutations are distributed throughout the domains of EphA3 (Figure 1). The frequency of EphA3 mutations in lung cancer is significantly higher than expected by chance, suggesting that EphA3 is a lung cancer “driver” gene, whose mutations play a causal role in cancer development and/ or progression.16 One study suggested that EphA3 is likely to be a protooncogene in lung cancer because it is a receptor tyrosine kinase and was found to be amplified in two lung
he Eph receptors represent the largest family of receptor tyrosine kinases and are divided into two classes: EphA and EphB. There are nine EphA receptors and five EphB receptors in the human genome.1,2 The EphA receptors prefer to bind the GPI-linked ephrin-A ligands, and the EphB receptors prefer to bind the transmembrane ephrin-B ligands, although interclass binding of ephrins to Eph receptors can occur. EphA and EphB receptors are both type I transmembrane proteins and have a similar multidomain structure. Their extracellular region contains an N-terminal ephrinbinding domain, a cysteine-rich region that can be further subdivided into a sushi domain and an epidermal growth factor (EGF)-like domain, and two fibronectin type III domains (Figure 1). The sushi domain, also known as the complement control protein (CCP) domain, contains approximately 60 amino acids arranged in a β-sandwich structure that is stabilized by two disulfide bonds (http://smart.embl-heidelberg.de/ smart/do_annotation.pl?DOMAIN=CCP). The intracellular region of the Eph receptors includes the juxtamembrane segment, the kinase domain, a sterile alpha motif (SAM) domain, and a C-terminal PDZ domain-binding motif. Ephrin binding induces Eph receptor signaling by promoting oligomerization in concert with several receptor−receptor interaction surfaces located in the ephrin-binding domain, sushi domain, N-terminal fibronectin type III domain, transmembrane domain, and SAM domain.2−7 The physical proximity of clustered Eph receptor molecules leads to their reciprocal transphosphorylation. Phosphorylation of several intracellular tyrosines enhances Eph kinase activity by disrupting the inhibitory interactions of the juxtamembrane segment with the kinase domain and causing partial ordering of the activation loop.5,8,9 Furthermore, some of the tyrosine-phosphorylated © 2012 American Chemical Society
Received: September 7, 2011 Revised: January 10, 2012 Published: January 14, 2012 1464
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Article
EphA3's ability to signal in response to ephrin ligands may facilitate the development and progression of several cancer types.
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EXPERIMENTAL PROCEDURES EphA3 Cloning and Mutagenesis. The full-length human EphA3 cDNA clone was purchased from Invitrogen (clone MGC:71556; GenBank accession number NP_005224.2) and subcloned into the pEGFP-IRES2 vector (Clontech, Mountain View, CA) between the EcoRI and BamHI restriction sites. The EphA3 mutants were generated using the QuikChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) following the manufacturer’s instructions. Wildtype and mutant EphA3 alkaline phosphatase (AP) fusion proteins were generated by subcloning cDNAs encoding wild-type and mutant extracellular domains (amino acids 1−537) into the pAPtag-2 vector (GenHunter, Nashville, TN) between BglII and BspEI restriction sites, except for the G228R mutant, which was cloned between the BamHI and BspEI restriction sites. Cell Lines and Transfection. HEK 293T cells were grown in Dulbecco’s modified Eagle's medium (Cellgro, Manassas, VA) supplemented with 10% fetal bovine serum, 1 mM L-glutamine, 1 mM sodium pyruvate, and antibiotics. Lipofectamine Plus (Invitrogen, Carlsbad, CA) was used to transfect HEK 293T cells with wild-type and mutant EphA3 cDNA in the pEGFP-IRES2 vector, following the manufacturer’s instructions and using various amounts of DNA (1−8 μg) to obtain similar wild-type and mutant EphA3 protein levels (see Figures 2A and 3B). Immunoblotting. For immunoblotting, cells were lysed in modified RIPA buffer [50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 2 mM EDTA] containing protease and phosphatase inhibitors. Cell lysates were separated by sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS−PAGE) followed by immunoblotting with an anti-EphA3 antibody (sc-919, Santa Cruz Biotechnology, Santa Cruz, CA) or an anti-human Fc antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) followed by either an anti-rabbit or an anti-mouse secondary antibody conjugated to horseradish peroxidase (HRP) or with a phosphotyrosine antibody conjugated to HRP (610012, BD Biosciences, San Jose CA). Immunoblots were developed with HyGLO chemiluminescence HRP detection reagent (Denville Scientific, Metuchen, NJ). Production of EphA3 AP. Ten micrograms of wild-type or mutant EphA3 AP plasmid DNA was transiently transfected into HEK 293T cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Culture medium containing secreted EphA3 AP fusion proteins was concentrated using Amicon Ultra-Centrifugal filters (Millipore, Billerica, MA). The concentration of the AP fusion proteins was estimated on the basis of AP activity measurements.26,27 Quantification of EphA3−Ephrin-A5 Binding by an Enzyme-Linked Immunosorbent Assay (ELISA). Protein A-coated 96-well plates (Pierce Scientific, Rockford, IL) were used to immobilize ephrin-A5 Fc (0.1 μg/mL) (R&D Systems, Minneapolis, MN) or Fc control (MP Biomedical, Solon, OH) for 1 h at room temperature in 3% BSA in Tris-buffered saline with 0.01% Tween 20 (TBST). Various amounts of culture medium containing concentrated EphA3 AP fusion proteins diluted in TBST were subsequently added for 1 h at room temperature. After three washes with TBST, binding was quantified using p-nitrophenyl phosphate (pNPP) (Pierce Scientific) as
Figure 1. Location of EphA3 somatic cancer mutations in the receptor domain structure. (A) Schematic illustrating the location of the studied mutations with respect to the domains of EphA3. Mutations identified in lung cancer are colored orange, mutations identified in other cancers black, and control mutations known to disrupt ephrin binding (T102Q) or kinase activity (K653R) blue. (B) Crystal structure of the extracellular domain of EphA2 bound to ephrin-A5 (PDB entry 2X11) used as a model to show the location of the EphA3 mutations because the structure of the extracellular region of EphA3 has not been determined. (C) Location of the mutations in the kinase domain of EphA3 (PDB entry 2QOQ). (D) Crystal structure of the SAM domain of EphA4 (PDB entry 1B0X) used as a model to show the location of the EphA3 mutations because the structure of the SAM domain of EphA3 has not been determined. The mutated amino acids are denoted by spheres, orange for mutations found in lung cancer, black for mutations found in other cancers, and blue for the two control mutations. A list of all somatic cancer mutations in EphA3 is provided in Table 1 of the Supporting Information.
cancer specimens.16 In addition, one of the mutations identified in the EphA3 kinase domain corresponds to an FGF receptor activating mutation. On the other hand, EphA3 was predicted to be a tumor suppressor in head and neck cancer, and the 3p11.2 chromosomal region where the EphA3 gene is located undergoes frequent loss of heterozygosity in different types of cancers.1,24 Characterization of the functional effects of the mutations found in lung cancer and other cancers could help resolve the role of EphA3 in these cancers. We therefore generated the 28 mutations that had been reported at the time our study began and systematically examined how they affect several aspects of EphA3 function. We found that many of the mutations disrupt EphA3 autophosphorylation and kinase activity, ephrin binding, and cellular trafficking. These results suggest that the loss of 1465
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Figure 2. Effects of mutations in the intracellular region of EphA3 on receptor tyrosine phosphorylation. (A) Representative immunoblots showing tyrosine phosphorylation (PTyr) and total levels of wild-type EphA3 and the indicated mutants expressed by transient transfection in HEK 293 cell lysates. The histogram shows averages ± SEM calculated from quantification of three separate experiments and normalized to the wild-type value in each experiment. (B) Representative immunoblots showing autophosphorylation of immunoprecipitated wild-type and mutant EphA3 after in vitro kinase assays with and without the addition of ATP. The histogram shows averages ± SEM calculated from quantification of three separate experiments and normalized to the wild-type value in each experiment. *P < 0.05 by Student’s paired two-tailed t test comparing phosphorylation of each mutant to that of the wild type. (C) Representative images showing phosphorylation of enolase by immunoprecipitated wild-type and mutant EphA3 as well as autophosphorylation after in vitro kinase assays with and without the addition of [γ-32P]ATP. The histograms show averages ± SEM calculated from quantification of three separate experiments and normalized to the wild-type value in each experiment. *P < 0.05 by Student’s paired two-tailed t test comparing mutants to the wild type. 1466
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the substrate. Dissociation constants (KD) were calculated using nonlinear regression and GraphPad Prism (Graphpad Software, Inc., La Jolla, CA). Assessment of EphA3−Ephrin-A5 Binding by a PullDown Assay. Transfected cells were lysed in modified RIPA buffer, and lysates were incubated with 1 μg of ephrin-A5 Fc immobilized on GammaBind Plus Sepharose beads (GE Healthcare, Piscataway, NJ). Complexes were washed three times with RIPA buffer, boiled in sample buffer, separated by SDS− PAGE, and probed by immunoblotting for EphA3. In Vitro Kinase Assay. HEK 293T cells expressing wildtype or mutant EphA3 were lysed in RIPA buffer without phosphatase inhibitors. EphA3 was immunoprecipitated with 2.5 μg of anti-EphA3 antibody (37-3200, Life Science TechnologiesInvitrogen, Carlsbad, CA) and immobilized on GammaBind Plus Sepharose. Immunoprecipitates were then incubated with 25 mM Hepes (pH 7.5), 10 mM MnCl2, 10 MgCl2, 1 mM sodium orthovanadate, 0.1% Triton X-100, and 150 μM ATP with phosphatase inhibitors for 30 min at 30 °C, and reactions were analyzed by immunoblotting for phosphotyrosine and EphA3. Immunoblots were quantified using Image J. To measure phosphorylation of enolase by EphA3, immunoprecipitates with 1.25 μg of anti-EphA3 antibody (Santa Cruz, sc-919) were washed twice with HNTG buffer [20 mM Hepes (pH 7.5), 150 mM NaCl, 10% glycerol, and 0.1% Triton X-100] and then twice with kinase reaction buffer [10 mM Hepes (pH 7.5), 25 mM MgCl2, and 10 mM MnCl2]. Rabbit muscle enolase (Sigma) was acid denatured, and 6.25 μg was added to each kinase reaction mixture together with 5 μCi of [γ-32P]ATP. 32P incorporated into enolase and EphA3 after incubation for 30 min at 30 °C was quantified from scans obtained with a Storm phosphorimager using Image J. Measurement of Ephrin-Binding Domain Folding. Protein-A plates were coated with 0.5 μg/mL anti-EphA3 antibody (sc-919, Santa Cruz Biotechnology) in 3% bovine serum albumin (BSA) in TBST to capture EphA3 from transiently transfected HEK 293T cells lysed in modified RIPA buffer. The amounts of cell lysates used were sufficient to saturate the binding sites of the coated anti-EphA3 antibody, as verified by comparing the results with those obtained with twice as much lysate (data not shown). This ensured that the same amount of EphA3 was immobilized in all the wells. EphA3 with a properly folded ephrin-binding domain was detected using 2 μg/mL IIIA4 anti-EphA3 antibody (from Millipore or a kind gift from A. Boyd) in 3% BSA in TBST, followed by a goat anti-mouse AP antibody (AP124A, Millipore), and binding was quantified using pNPP as the substrate. Quantification of Cell Surface EphA3. HEK 293T cells expressing wild-type or mutant EphA3 were incubated with EZ-link SulfoNHS-LC-Biotin (Pierce-Thermo Scientific, Rockford, IL) to biotinylate cell surface proteins. Lysates were captured using an anti-EphA3 antibody recognizing an intracellular epitope (Santa Cruz; see the previous section), and biotin labeling was detected using a streptavidin−HRP conjugate (PierceThermo Scientific). The amounts of cell lysates used were sufficient to saturate the binding sites of the coated antiEphA3 antibody, as verified by comparing the results with those obtained with twice as much lysate (data not shown). This ensured that the same amount of EphA3 was immobilized in all the wells.
Article
RESULTS
Mutations in the EphA3 Intracellular Region Affect Tyrosine Phosphorylation and Kinase Activity. Phosphorylation of cytoplasmic tyrosine residues is a hallmark of Eph receptor activation and downstream signaling.3 Like other Eph receptors, wild-type EphA3 is constitutively tyrosine phosphorylated when overexpressed by transient transfection in HEK 293 cells (Figure 2A).28−30 Interestingly, we found that four of the eight mutations examined in the EphA3 kinase domain [R728L, K761N, G766E, and D806N (Figure 1)] essentially abolish EphA3 tyrosine phosphorylation in HEK 293 cells (Figure 2A and Table 1). This effect is similar to that of Table 1. Effects of EphA3 Mutations1
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Legend: =, similar to that of the wild type;
